Behavioral and physiological responses of dairy goats to isolation

Behavioral and physiological responses of dairy goats to isolation

Physiology& Behavior,Vol. 51, pp. 297-301. ©Pergamon Press plc. 1992. Printed in the U.S.A. 0031-9384/92 $5.00 + .00 Behavioral and Physiological Re...

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Physiology& Behavior,Vol. 51, pp. 297-301. ©Pergamon Press plc. 1992. Printed in the U.S.A.

0031-9384/92 $5.00 + .00

Behavioral and Physiological Responses of Dairy Goats to Isolation I D. A. CARBONARO, 2 T. H. FRIEND, G. R. D E L L M E I E R

Department of Animal Science, Texas A & M University, College Station, TX 77843 AND L. C. N U T I

International Dairy Goat Center, Prairie View A&M, Prairie View, TX 77446 R e c e i v e d 11 June 1990 CARBONARO, D. A., T. H. FRIEND, G. R. DELLMEIER AND L. C. NUTI. Behavioral and physiological responses of dairy goats to isolation. PHYSIOL BEHAV 51(2) 297-301, 1992.--Eight Nubian and eight Alpine dairy goat does were used in a crossover experimental design to determine the effect of 30 min of isolation on behavior and plasma concentrations of thyroxine (T4), triiodothyronine (T3), cortisol, norepinephrine (NOR), and epinephrine (EPI). Isolation was hypothesized to produce an emotional state analogous to fear. Focal animal behavior was recorded for the initial five min of isolation. Blood samples were obtained via jugular cannulae at 0 (immediately prior), 10, 20, 30 (during isolation), 40, 50 and 60 min (after return to their group). Response to isolation was characterized physiologically by increased plasma concentrations of NOR (p<0.01), but not T 3, Ta, cortisol or EPI, indicating a sympathetic discharge. Isolated goats also vocalized more frequently (p<0.01) and spent a greater amount of time sniffing, trotting and rearing (p<0.05). The Nubian does reacted more strongly (elevated NOR, trotting and rearing, p<0.01) to isolation than the Apline does. Goats

Emotions

Fear

Thyroxine

Catecholamines

THERE is a need for a formal, scientific assesment of the emotional, or more precisely, behavioral states in livestock (15). Being able to differentiate physiologically between general physical stressors such as voluntary exercise, versus adverse psychological stressors such as fear, is central to answering concerns about animal welfare. Livestock producers are also concerned because stress in livestock results in decreased reproduction and productivity as well as an increased susceptibility to disease (7, 15, 19). Mason (13) proposed that psychological factors play a decisive role in activating the neuroendocrine system. In his many studies of rhesus monkeys exposed to different types of physiological and/or psychological stressors including avoidance conditioning, cold and fasting, patterns of the hormonal response differed from one type of stressor to another. Mason proposed that the pituitary-adrenalcortical response may actually be a specific reaction for psychological stress rather than a nonspecific reaction for all stressors as Selye (19) had proposed. A similar situation has been shown in livestock. For example, when sheep were exposed to a variety of stressors, i.e., shearing, yarding, estrogen administration, feeding and fasting, increases in plasma cortisol were observed. However, the pattern of release differed for each stressor (10). The quantification of catecholamine re-

Cortisol

lease can also be used as indicators of well-being as well as a means of differentiating between various emotional or behavioral states (7,20). For example, public speaking will cause a fourfold increase in epinephrine (adrenal response) in medical students, whereas having them walk up and down flights of stairs causes the reverse to occur (3). The possibility of using catecholamines to differentiate between different types of stressors has not been investigated in livestock. The objective of this study was to characterize changes in the behavior and physiology of goats in a state of fear induced by isolation. Subjecting sheep and goats to temporary isolation is an accepted procedure to induce a state of fear (17). This and a companion study (2) were designed to contribute toward developing the techniques and methodology necessary to objectively assess the various behavioral states in livestock. METHOD

Animal Management In Experiment I, eight open Nubian does were randomly assigned to two groups ( N = 4 ) . Each group of four does was housed for one week in a 3.0 x 4.4 m environmental chamber.

1Technical article No. 25671 from the Texas Agricultural Experiment Station. 2Present address: Division of Orthopedic Surgery, Children's Hospital of Los Angeles, Los Angeles, CA 90054-0700.

297

298

The environmental chamber prevented any audio, olfactory or visual contact with any other goats. The goats were fed a maintenance diet with water available ad lib. The four does were randomly allocated to two groups of pairs. On day 8 after baseline blood samples were obtained from one of the pairs of does, one of the does from that pair was removed from the group and isolated for 30 min in an identical environmental chamber. The remaining member of that pair served as a control. After 30 min the isolated doe was returned to the group. Approximately one hour later baseline blood samples were obtained from the other pair of does. One of that pair was then similarly isolated for 30 min and then returned to the group. On the following day at the same time the protocol was repeated, with the goats being crossed over to the opposite treatment. Experiment II utilized the identical protocol for eight open Alpine does. Due to the availability of only two environmental chambers, each trial (group of four does) was tested consecutively. The does were part of Prairie View A & M ' s International Dairy Goat Center milking herd and were completely habituated to the presence of humans and most routine activities. Although researches entered the chambers at 10 min intervals to draw blood samples, the control does showed little reaction to the researchers who scratched or petted the does while obtaining the samples. Similarly, the other activities such as returning the isolated does to the group had only a minimal effect on the control does. The isolated does did direct their behavior toward the researcher as soon as their chamber was entered. Because the isolated does did approach the researcher, the blood sample was usually obtained in less then one min and the doe was again in isolation. In a preliminary experiment, the control and isolated does were maintained in metabolism crates within the chambers. The crates restricted the movement of the does so that blood samples could be obtained through long cannulae without entering the chamber. However, this experiment was abandoned because of the confounding that occurred from the stress associated with restricting the movement of the does by using the crates, as was found for calves confined in similar crates (81).

Physiological Data Collection In order to minimize stress due to repeated blood sampling, jugular vein cannulae were established in each pair of does one hour prior to testing. Lower concentrations of plasma catecholamines were obtained from rats not disturbed by handling and restraint due to the use of an indwelling arteriol catheter (16). Plasma cortisol returned to normal concentrations within one hour after cannulation of swine (9). The cannulae consisted of 18-gauge medical grade tubing held in place with adhesive tape. Disodium ethylenediamine tetraacetate (EDTA) (20 mg/ml) was placed into the tubing to prevent clotting between blood sampies. A nonpalatable agent, Chew Guard ®, was applied to the wrapping to discourage nibbling and eating of the wrapping by other does. The cannulae remained in the goats over night and were used when the treatments were reversed the second day. Because only minimal restraint of the goats was required during cannulation (a second person gently holding the goat so she would not walk off), and because of the reversal of treatments on the second day, any effect from cannulation should be minimal and equal across treatments. Blood samples were drawn simultaneously from the isolated and control doe immediately prior to isolation (time 0), during isolation (10, 20 and 30 min), and after return to the group (40, 50, and 60 min). Each sample was obtained with a 10 cc sytinge containing 10 mg ETDA as an anticoagulant. The samples were transferred to glass tubes and maintained in an ice bath un-

CARBONARO. FRIEND DEI.LMEIER ANI) N{;II

TABLE OPERATIONAL DEFINITION OF BEHAVIORS RECORDED

Defecation: Eat: Groom: Headmovement: Kneel: Mouthing: Paw: Recumbancy: Sniff: Stand: Rear: Trotting: Urination: Vocalization: Walking:

Elimination of feces Mastication, swallowing and/or rumination of feed Licking, chewing or rubbing of one's body parts Change in head rotation >90 ° on the x and/or z axis and/or elevation ~90 ° on the y axis supporting the body with the knee of the forelegs and feet of extended hindlegs Manipulation of an object with one's mouth Forward, striking motion made with foreleg; may or may not strike the ground Lying laterally or sternally Gesture directing the nose within 10 cm of a surface and/or object Supporting the body with all four legs extended with no significant forward or backward locomotion Jumping or standing up to support the body with the hindlimbs and a vertical surface Form of forward locomotion; two beat gait on alternating diagonal legs Elimination of urine All sounds made with the vocal cords, generally bleats and screams Form of forward locomotion; a slow four beat gait

til centrifugation. The plasma was drawn off into duplicate polypropylene tubes. For the determination of the catecholamines epinephrine (EPI) and norepinephrine (NOR), equal volumes of plasma and perchloric acid (0.1 M) were added to one tube. The perchloric acid deproteinates the plasma, allowing the sample to be stablized for storage. The samples were capped and frozen until analysis could be completed.

Behavioral Data Collection Behavioral data were collected during the first five min after the initiation of isolation. Simultaneous focal-animal (1) observations were made for the control and isolated animals. Observations were made through a small observation window located in the door of each chamber. All behavioral data were collected simultaneously by the same two observers who rotated treatments. The goats did not appear to be aware of the presence of people during the testing procedure. All observations were dictated into a tape recorder. The tapes were later replayed and each behavior was timed with a stop watch. The events and states recorded are summarized in Table l along with the operational definition of each behavior. Vocalizations were also given a subjective score of intensity: 1 was barely audible; 2 was a normal, audible bleat; and 3 was a very, loud, scream-like vocalization.

Hormone Determinations The catecholamine extraction procedure and assay used was adapted and modified from an application note furnished by Bioanalytical Systems Inc., West Lafayette, IN, and described in a companion paper (2). Seventeen percent of the samples analyzed for NOR had concentrations below the lower detectable threshold of 100 pg/ml and were, therefore, assigned a default value of 100 pg/ml. Fifty-six percent of the samples analyzed for EPI had concentrations below the lower detectable threshold

RESPONSE OF GOATS TO ISOLATION

299

TABLE 2 LEAST-SQUARES MEANS OF BEHAVIORAL DATA

e. Treatment Behavior Frequency (5 min): Paw Head Movement Vocalizations Intensity

Control (n = 14)

-0.04 5.84 2.63 0.35

+__0.16 ± 1.42 ± 5.13 ± 0.14

Percentage of time (5 min): Mouthing 0.93 ± 0.53 Self Groom 1.44 ± 0.55 Sniffing 1.69 - 2.54 Recumbent 0.86 --- 3.47 Standing 92.32 ± 3.43* Kneeling - 0 . 0 4 -+ 0.26 Walking 8.07 ± 2.21 Trotting 0.27 + 0.28 Rearing -0.08 ± 2.14 Eating 19.38 ± 9.99

i

|

Isolated (n = 14)

0.40 0.06 44.03 1.90

± ± ± ±

0.17 1.56 5.64* 0.15"

0.02 0.06 13.16 3.33 70.17 0.40 13.34 1.45 9.68 25.41

± 0.58 ± 0.61 ± 2.80t ± 3.82 ± 3.78 ± 0.28 _ 2.44 ± 0.36t ± 2.36~ ± 10.99

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.......~

0

10

~

o,~ N

*Significantly higher than other values in row (p<0.1). tSignificantly higher than other values in row (p<0.5).

of 80 pg/ml and were, therefore, assigned a default value of 80 pg/ml. Because of the large percentage of values below the detectable threshold these data should be interpreted with caution because this is a conservative approach to handling such data. Cortisol, triiodothyronine (T3) and thyroxine (T~) concentrations were determined by double antibody iodinated kits (Pantex, Santa Monica, CA). All determinations were made on duplicate samples with a lower sensitivity of 0.3 ng/dl for cortisol, 25 ng/dl for triiodothyronine and 0.4 Ixg/dl for thyroxine. The within- and between-assay coefficients of variation from a pooled sample for cortisol were 8.7 and 9.3%; for T a were 2.1 and 2.3%; and for thyroxine were 3.3 and 4.8%. All plasma hormone responses were quantified by integrating the area under the curve described by the plasma levels from 5 to 60 minutes by: (H 2 + H~+~)/2 × I, where H is the hormone concentration in plasma of sample s and the successive sample, s + 1, with an interval of I hours between the samples (8). Three missing data points (2 for one animal) were encountered for each breed because of cannulae failure. Missing data points were estimated by averaging two averaged values. The first value was the average across all sampling times within that goat and the second value was the average of that particular variable from goats of the same breed and treatment at that particular sampling time.

E i[

!

E

ia~ LU

Z

20

30

40

50

60

TIM[(mln)

Statistical Analysis General Linear Model (18) tests for the main effects of treatment, breed, their interaction, as well as for the nested effects of breed, replication, trial and goat were conducted. Two analyses were conducted. In one, the baseline plasma concentrations for each hormone (time 0) was used as a covariate. In the other analysis no covariate was used. The only difference between the analyses was that the use of the corvariate increased the level of significance by one or two units for the analyses in which the

FIG. 1. Hormone profiles of Nubian and Alpine does maintained in a group (control) or removed from the group and isolated from time 0-30 min (shaded area). The isolated does (N = 8 per breed), were returned to their group of four does immediately after the 30-min blood sample was obtained.

covariate was significant. Therefore, only the results from the analysis with the covariate are reported. There were several occasions in which working cannulae could not be established so

300

CARBONARO, FRIEND, DELLMEIER :\ND NUI'i

TABLE 3 LEAST-SQUARESMEANS ( _+SE) OF INTEGRATEDHORMONE RESPONSESFOR PLASMASAMPLESOBTAINEDDURINGISOLATION (0-30 MIN) AND AFTERRETURNTO GROUP (40-60 MINI POOLED FOR BOTHNUBIANAND ALPINEDOES Treatment Control (n = 14)

Hormone Thyroxine, Ixg/dl/h 0-30 min samples* 40-60 min samplest:~ Triiodothyronine, ng/ml/h 0-30 min samples 40--60 min samples Cortisol, p,g/dl/h 0-30 min samples 40-60 min samples Norepinephrine, pg/ml/h 0-30 min samples§ 404~0 min samples Epinephrine, pg/ml/h 0-30 min samples* 40-60 min samples

4.62 +_ 0.51 5.01 _+ 0.24 39.3 39.1

_+ 1.7 + 2.4

1.39 + 0.24 1.47 + 0.18

Isolated (n = 14)

4.5 -+ 0.52 5.44 +- 0.24 41.0 38.6

_ 1.8 +_+_ 2.4t

1.33 --- 0.24 1.42 - 0.18

149.9 --+ 23.6 167.0 -+ 27.5

241.5 260.6

186.5 163.8

194.0 +_ 4.7 227.8 _+ 29.2

- 4.7 --_ 29.1

+_ 23.7# +_ 27.6¶

*Baseline sample was significant as a covariate (0<0.05). 1-Trial (breed * replication) effect (0<0.05). z~Treatment * breed effect (0<0.01) with nubians higher in control. §Higher in Nubians in both treatments (.o<0.05). #Higher than other value in row (0<0.03) using t-test with adjusted variance. ¶Higher than other value in row (0<0.06) using t-test with adjusted variance.

least-squares means (population marginal means) were used to approximate the values for a balanced design. Because a relatively high percentage of the NOR and EPI concentrations were below the lower sensitivity of the assay and the variation for those samples was artificially reduced by assigning the value of the lower detectable threshold (LDT), NOR and EPI treatment effects were determined using a t-test in which the variance was first calculated by assigning zero to all samples at the LDT. This inflated variance was then applied to a t-test in which LDT values were used to calculate the means and determine treatment effects. RESULTSAND DISCUSSION

Behavioral Characteristics When the does were subjected to isolation, they displayed an increase in the frequency and intensity of vocalizations (Table 2). Isolated does also spent a greater percentage of time sniffing surrounding objects and trotting about the chamber and rearing upon the stainless steel walls. In contrast, the control animals spent a greater amount of time standing still. There were no incidents of elimination behaviors. Increases in vocalizations and general activity were also observed by Price and Thos (17) in isolated goats and sheep. Moberg et al. (14) also found that when Iambs were placed alone in a novel environment there was an increase in vocalization and in the percentage of time spent moving. Similarly, piglets isolated in an unfamiliar pen greatly increased certain types of vocalizations (6).

In this experiment, the Nubian does were more affected h,, isolation: isolated Nubian does vocalized more (p<0.(lt~ than isolated Alpine does, 52.4_+ 0.6 versus 35.7 := 7.2, rcspectivel?. Also, isolated Nubians spent more time trotting i2.~: + 3.7% verus 0.28-+0.4%, p<0.05) around the environmental chamber. and rearing up on the walls (12.8-+-0.47% versus 6 5 4 + ~.0%. p<0.05).

Physiological Characteristics Plasma NOR response was significantly higher for the isolated goats during isolation (p<0.03) and after return to the group (p<0.06, Table 3). There were no significant treatment differences for T 4, T 3, cortisol or EPI, although EPI was slightly elevated. The elevated NOR data suggests there was an overall sympathetic discharge with little adrenal medullary stimulation (13). Sympathetic discharge or NOR release has been mainly associated with exercise and physical stressors, whereas EPI or adrenal medullary release has been found to occur largely in tesponse to psychological stressors (3, 4, I1). In this study, the greatly increased physical activity displayed by the isolated does may have masked the differential release of NOR and EPI that is indicative of psychological stress. T 3, T 4 and cortisol responses did not change with isolation, in contrast to reported (5) increases in circulating thyroid hormones when sheep were either subjected to restraint, cannulation, a barking dog, or loud explosions. Norepinephine response (Table 3) was significantly greater (adjusted t-test) for does during isolation (p<0.03) and during the 30-min period following return to the group (p<0.06) while EPI was not significantly altered. Such a trend would indicate that a physical and not an emotional response was predominant (3). This is consistent with the large amount of physical activity displayed by the isolated does (Table 2), that may have masked the emotional component. Causual observation of Fig. 1 indicates a trend for T 4, T 3 and NOR to return to pretreatment concentrations by the end of the sampling period (60 min). The tendency for Nubians to be more responsive in general than Alpines is also evident in T 4 and NOR. The hormone that was the most responsive to treatments was clearly NOR.

Behavioral and Physiological Correlations No significant correlations were found between the behavioral and physiological characters that were measured. This was not expected and may have been due to the physical activity the does displayed during isolation acting as a coping mechanism. CONCLUSIONS The isolated does in this study exhibited similar hormonal profiles to goats subjected to food thwarting (2). This finding is supported by others who concluded that NOR release is highly associated with exercise or muscular activity (3, 11, 12). This increase in NOR probably results from sympathetic discharge because the adrenal medulla's contribution to plasma concentrations of NOR is relatively small (11,12). The physiological characteristics measured in this and the companion food-thwarting study (2) indicated that there was arousal, but they could not be used to differentiate between isolation induced fear and food thwarting (frustration). The behavioral data, however, were relatively distinctive. Although the frustrated and isolated does displayed signs of arousal, the isolated does vocalized frequently, whereas the frustrated does pawed and performed many head movements.

R E S P O N S E O F G O A T S TO I S O L A T I O N

301

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11. Goldstein, D. S.; McCarty, R.; Polinsky, R. J.; Kopin, I. J. Relationship between plasma norepinephrine and sympathetic neural activity. Hypertension 5:552-559; 1983. 12. McCarty, R.; Kopin, I. J. Plasma catecholamines and behavior of rats during stress. In: Usdin, E.; Kopin, I. K.; Barchas, J. D., eds. Catecholamines: Basic and chemical frontiers. New York: Pergamon Press; 1789-1791; 1979. 13. Mason, J. W. A reevaluation of the concept of "non-specificity" in the stress theory. J. Psychiatr. Res. 8:323-333; 1971. 14. Moberg, G. P.; Anderson, C. O.; Underwood, T. R. Ontogeny of the adrenal and behavioral responses of lambs to emotional stress. J. Anim. Sci. 51:138-142; 1980. 15. Moberg, G. P. A model for assessing the impact of behavioral stress on domestic animals. J. Anim. Sci. 65:1228-1235; 1987. 16. Popper, C. W.; Chieuh, C. C.; Kopin, I. J. Plasma catecholamine concentrations in unanesthesized rats during sleep, wakefulness, immobilization and after decapitation. J. Pharmacol. Exp. Ther. 202: 144-148; 1977. 17. Price, E. O.; Thos J. Behavioral responses to short-term social isolation in sheep and goats. Appl. Anim. Ethol. 6:331-339; 1980. 18. SAS Institute, Inc. SAS guide for personal computers. Cary, NC: Statistical Analysis Institute, Inc.; 1985. 19. Selye, H. Stress and disease. Science 122:625-631; 1955. 20. Stott, G. H. What is animal stress and how is it measured? J. Anim. Sci. 52:150-153; 1981.